This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. This investigation will focus on the role and regulation of actin and myosin remodeling in the contracting A7r5 smooth muscle cell in comparison to aortic smooth muscle tissue. We have previously provided evidence of differential remodeling of the alpha- and beta- actin as well as smooth muscle myosin II cytoskeletal structures. However, the mechanism(s) regulating this differential remodeling is not understood. Data suggests that the redistribution of actin- actin to podosomes is more sensitive to phorbol stimulation than redistribution of beta-actin to the podosomes. Myosin distribution appears to follow changes in alpha-actin component of the cytoskeleton. Also, preliminary evidence suggests that Rho kinase inhibition, previously shown to antagonize the development and maintenance of contraction in rat aortic smooth muscle tissue, blocked alpha-actin remodeling to the podosomes and re-establishment of alpha-actin stress cables. This evidence supports the hypothesis of differential remodeling and potentially different regulatory mechanisms governing the actin cytoskeleton. The major objective of the proposed project is to determine the effects of Rho kinase inhibition on actin and myosin remodeling in A7r5 cells and compare this to the effect previously observed in aortic smooth muscle tissue. Preliminary evidence suggests several Rho kinase may have a selective effect on alpha-actin and therefore support our hypothesis of differential remodeling of the alpha-actin and beta-actin cytoskeleton. This would suggest that there may be several possible biochemical pathways regulating remodeling and therefore contraction in smooth muscle. If kinase regulation of cytoskeleton is differentially regulated, it would support our model of contraction which assigns different roles to alpha- and beta-actin during smooth muscle contraction. Also, over the two year funding period, this project will engage numerous undergraduate and graduate students in research, enhancing their understanding of the scientific process and allowing them to make contributions to a poorly understood physiological mechanism.